Poxviruses encode enzymes and factors needed for transcription and replication of their genomes within the cytoplasm of infected cells. Vaccinia virus, the prototypic member of the poxvirus family, provides a unique system for combining biochemical and genetic approaches for investigating mechanisms of gene regulation, mRNA biosynthesis, and DNA replication. Studies with vaccinia virus indicated that the genes are divided into three temporal classes - early, intermediate and late. Each gene class has a consensus DNA promoter sequence and corresponding transcription factors that interact with the virus-encoded multisubunit RNA polymerase. The transcription system for early genes is packaged within the infectious virus particle during its assembly, whereas the factors for intermediate and late gene transcription are synthesized successively after infection.The DNA is synthesized as concatemers that are resolved into unit length genomes and packaged during virus assembly. All poxviruses have a highly conserved DNA topoisomerase that is a potential target of antiviral drugs. Although the structure and catalytic activity of the enzyme were well studied, little was known about its biological function. The viral topoisomerase was thought to be essential; and roles in DNA replication, recombination, concatemer resolution and transcription were suggested. We have now demonstrated that the topoisomerase is not essential for replication of vaccinia virus in cultured cells, although deletion mutants formed fewer and smaller plaques on cell monolayers than wild type virus. Purified mutant virus particles were able to bind and enter cells, but exhibited reduced viral early transcription and a delay in DNA replication. The data suggest that the primary, perhaps only, role of the poxvirus topoisomerase is to increase early transcription, which takes place within virus cores in the cytoplasm of infected cells. Because the topoisomerase functions early in infection, drugs capable of penetrating the virus core and irreversibly damaging DNA by trapping nicked DNA-topoisomerase intermediates could make potent antiviral agents.